Abstract The significant amount of data generated by SIV vaccine studies has led to the suggestion that an HIV vaccine is achievable. The capacity of humoral and cellular immune responses in mucosal tissues to block or contain replication at the initial stage of virus transmission may have a profound impact on the ability of a vaccinated host to resist infection. The development of an effective vaccine that restricts viral replication at the mucosal portal of entry may be our best hope for controlling HIV infection. We believe there are two necessary features for a successful vaccine: 1) life-long stimulation of the immune system with viral antigens; and 2) a targeted immune response at the site of primary replication of HIV. A vaccine approach that simultaneously addresses these two issues would have the potential to achieve solid, long-term protection. To fulfill these requirements, we propose an alternative approach to successfully deliver a vaccine to mucosal sites and elicit protective mucosal immune responses. We propose to use the epithelial stem cell as a permanent source of viral antigen and their differentiated offspring as antigen producing presenting cells. Using a single cycle SIV (SIVsc) approach, which has been shown to be a very safe strategy compared to traditional attenuated vaccines, we propose to clone the SIVsc genome under the control of the involucrin promoter, a terminally differentiated keratinocyte specific promoter. This virus will be then administered to target epithelial stem cells from different tissues (epidermal, vaginal, rectal). Basal layer cells will divide and differentiate thus triggering SIV antigen expression and both direct and cross priming. Herein, we propose: 1) To elicit SIV antigen expression from a terminally differentiated keratinocyte- specific promoter in rhesus macaques vaccinated by dermal, vaginal and rectal routes;2) To investigate the nature of immune responses induced by the different inoculation protocols;and, 3) if these animals are able to mount a satisfactory immune response compared to attenuated SIV infected animals, we will attempt to demonstrate protection against multiple low-dose vaginal and rectal challenges with a homologous SIV strain.